Abstract
Fluorescence in situ hybridization (FISH) is the primary technology used to image and count mRNA in single cells, but applications of the technique are limited by photophysical shortcomings of organic dyes. Inorganic quantum dots (QDs) can overcome these problems but years of development have not yielded viable QD-FISH probes. Here we report that macromolecular size thresholds limit mRNA labeling in cells, and that a new generation of compact QDs produces accurate mRNA counts. Compared with dyes, compact QD probes provide exceptional photostability and more robust transcript quantification due to enhanced brightness. New spectrally engineered QDs also allow quantification of multiple distinct mRNA transcripts at the single-molecule level in individual cells. We expect that QD-FISH will particularly benefit high-resolution gene expression studies in three dimensional biological specimens for which quantification and multiplexing are major challenges.
Highlights
Fluorescence in situ hybridization (FISH) is the primary technology used to image and count mRNA in single cells, but applications of the technique are limited by photophysical shortcomings of organic dyes
We show that quantum dots (QDs)-FISH provides improved signal stability, improved fidelity of molecular counting, and the capacity for multiplexed RNA quantification at the single-molecule level
We generated a series of QDs coated with multidentate polymers that allow the total hydrodynamic diameter of the probe to be as small as ~7 nm
Summary
Fluorescence in situ hybridization (FISH) is the primary technology used to image and count mRNA in single cells, but applications of the technique are limited by photophysical shortcomings of organic dyes. Considerable industry investment, and broad use in solution-based assays, QDs have not been widely used in FISH protocols This is due to inaccurate labeling resulting from the large sizes (15–35 nm) of commercially available probes[11,12], which cannot transport into crowded macromolecular environments of fixed cells to densely label targets. Hydrodynamic diameters of the respective aqueous QDs were 9.2 nm (QD9.2), 13.3 nm (QD13.3), and 17.4 nm (QD17.4) measured by protein-calibrated gel permeation chromatography (GPC, Fig. 1c) Both QD9.2 and QD13.3 yielded GAPDH mRNA counts that were similar to those of dyes (p > 0.05; Student’s t-test), whereas counts using QD17.4 labels were significantly lower (p < 0.001). An alternative large QD variant from a commercial vendor (QDcom) with dissimilar surface chemistry (PEG-coated amphiphilic polymers) likewise under-labeled RNA targets (p < 0.001)
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